The pyrazolopyrimidine zaleplon is a short-acting, benzodiazepine-like sedative/hypnotic used for the treatment of insomnia (Elie et al 1999). Following oral administration it is rapidly absorbed, the blood concentration peaking after approximately one hour (UKMI monograph 2000). It has a half-life of approximately one hour and is primarily metabolized by aldehyde oxidase and to a lesser extent by CYP3A4, the majority of a single dose being eliminated in urine as 5-oxozaleplon, 5-oxo-N-desethylzaleplon and 5-oxozaleplon glucuronide, while less than 1% is excreted unchanged. The closely related analogue, indiplon, has similar properties. Because of its rapid action and short half-life, zaleplon is increasingly finding use in drug-facilitated crimes (e.g. robbery, sexual assault, mugging) and recreational abuse, hence methods are required for the detection of zaleplon and indiplon. Horstkötter et al. (2003) described a capillary electrophoresis method for detection of zaleplon and metabolites in urine following solid-phase extraction, with detection limits of 10 ng/ml for zaleplon and N-desethylzaleplon and 100 ng/ml for 5-oxozaleplon and 5-oxo-N-desethylzaleplon Zhang et al. (2006) described a HPLC-MS method for detection of zaleplon in human plasma, with a detection limit of 0.1 ng/ml.
Specific binding reactions, such as antibody-antigen interactions, have been used extensively in immunoassays to detect a variety of substances present in biological samples. Compared to methods such as HPLC and LC-MS, such methods are less costly, require non-specialist staff for implementation and can be applied outside of the laboratory e.g. incorporated into a dipstick device. Thus, radioimmunoassays (RIAs) could be used for the determination of zaleplon, indiplon and their metabolites. Radioimmunoassays are very sensitive, but do require radionuclide tracers, for example 125I and 3H. Enzyme-linked immunosorbent assays (ELISAs) are a non-radioactive alternative that could be used for their qualitative and quantitative determination. There are no known RIAs or ELISAs for zaleplon, indiplon or their metabolites.
Immunoassays for the sensitive and specific determination of zaleplon and/or indiplon are desirable for application in therapeutic, toxicological and clinical settings. The invention described herein reports for the first time the development of two highly sensitive polyclonal antibodies for the detection of zaleplon, its metabolites and indiplon. The invention further describes methods and kits for the detection of zaleplon, its metabolites and the related pyrazolopyrimidine indiplon.